This proposal focuses on defining at a mechanistic level, the role in erythropoiesis of a newly discovered gene, HERF1, that we cloned as a downstream target of the AML1/CBFbeta transcription factor complex. The HERF1 protein contains an N-terminal RING-finger domain followed by a single B-box motif, a leucine coiled-coil domain, and a C-terminal rfp homologous region. Expression of HERF1 during embryogenesis coincides with the appearance of definitive erythropoiesis and, in adult mice, is restricted to the erythroid lineage, increasing over 40-fold during terminal differentiation. Importantly, we have demonstrated that inhibition of HERF1 expression blocks terminal erythroid differentiation, whereas over-expression of HERF1 in erythroid cells induces beta-major globin expression and erythroid differentiation. Taken together, these results suggest that HERFF1 plays an essential role in the development of mature erythroid cells. The studies in this project seek to elucidate, at a mechanistic level, the role of HERF1 in the linage commitment and maturation of erythroid progenitors. Our working hypothesis is that HERF1 functions as a part of a multiprotein complex to regulate cellular pathways required for the development and terminal differentiation of erythroid cells. Experiments are proposed in specific aim 1 to examine the biologic role of HERF1 in erythroid development through generation of both HERF1-deficient ES cells and mice, as well as murine systems with enforced HERF1 expression. In experiments outlined in specific aim 2, we will investigate the biochemical mechanism of action of HERF1 to gain insight into its mechanistic role in erythroid cell development. Together, these studies should provide important information on critical regulatory pathways required for the normal development of cells of the erythroid lineage. This fundamental knowledge is likely to expand our abilities to rationally manipulate erythroid lineage functions, potentially for therapeutic purposes in sickle cell anemia